A label-free strategy for measuring the affinity between monoclonal antibody and hapten using microdialysis sampling...

It is of great importance to measure antibody affinity in the course of screening monoclonal antibody (McAb) for immunotherapy, immunoassay and immunological purification. Herein, by using terbutaline mouse McAb as a model, a novel label-free strategy based on on-line microdialysis (MD) sampling combined with flow injection chemiluminescent detection was designed for measuring antibody affinity to hapten in a homogeneous system. After this McAb incubated with its hapten, the unbound hapten was sampled on-line by the MD probe and injected into the chemiluminescent detection system for quantification. The obtained concentrations of the unbound hapten were treated with Scatchard analysis and Klotz analysis to calculate the affinity constant. The MD probe showed a recovery of 26.2% for terbutaline under the chosen conditions. The affinity constants obtained using Scatchard analysis and Klotz analysis were 4.9×10⁶M^-1 and 4.9×10⁶M^-1, respectively, showing negligible difference. The obtained affinity constants indicated that the investigated McAb was an antibody with medium affinity. The designed strategy provided a simple, rapid and low-cost approach for direct measurement of antibody affinity. Furthermore, it avoided the decrease of affinity, which was encountered frequently in the conventional approaches based on probe labeling of McAb and protein conjugation of hapten.